Antitumor agents with the use of hsv

ABSTRACT

It is intended to provide highly safe antitumor agents which exhibit an antitumor effect on human remote tumors such as metastatic tumors too and by which an antitumor immune reaction enabling an immune therapy for cancer can be induced, tumor immunity inducers, T cell activators, dendritic cell activators, a method of treating cancer using the same, etc. Inactivated herpes simplex virus (inactivated HSV), herpes simplex virus glycoprotein D (HSVgD), etc. are employed as the active ingredients of antitumor agents, tumor immunity inducers, T cell activators or dendritic cell activators. As a specific example of the treatment for the above-described inactivation, citation may be made of a combination of UV-irradiation using ultraviolet light at 254 nm at 4 J/m 2  for 30 minutes with heating at 56° C. for 30 minutes.

TECHNICAL FIELD

The present invention relates to antitumor agents, tumor immunityinducers, T cell activators, and dendritic cell activators that exhibitantitumor effects on human distant tumors such as metastatic tumors aswell, and that contain an inactivated herpes simplex virus (inactivatedHSV) or herpes simplex virus glycoprotein D (HSVgD) etc. as an effectiveingredient. The present invention also relates to enhancement ofantitumor effects, methods for treating distant tumors such asmetastatic tumors, methods for inducing tumor immunity, methods foridentifying tumor antigens, methods for activating T cells, and methodsfor activating dendritic cells based on the use of the above-mentionedagents, inducers, or activators.

BACKGROUND ART

Induction of tumor-specific immunity enables long-term prevention ofrecurrence of tumors. Such immunotherapy, however, basically depends onthe presence or absence of tumor specific antigens and on whether or nota cytotoxic immune response can be induced, in which an antigen ispresented and tumor cells are recognized. Cytotoxic T lymphocytes(CTLs), together with costimulatory molecules, recognize MHC class Imolecules complexed with peptides derived from cytoplasmic proteinspresented on the cell surface (refer to e.g., non-patent literature 1).Tumor-specific antigens have been detected in various human tumors(refer to e.g., non-patent literatures 2and 3). Cancer vaccine therapyhas focused either on the use of inactivated tumor cells or theirlysates administered together with an adjuvant or a cytokine. It wasrecently reported that gene transfer of various cytokines, MHCmolecules, costimulatory molecules, or tumor antigens to tumor cellsenhances the visibility of tumor cells to immune effector cells (referto e.g., non-patent literature 4). If induction of antitumor immunitybecomes possible by direct intratumoral inoculation (in situ cancervaccine) of HSV etc, major problems in using cancer vaccines fortherapeutic purposes will be overcome. That is, the harvest of patients'autologous tumor cells, their in vitro manipulation such as culture andirradiation and identification of specific tumor antigens will not beneeded for preparation of cancer vaccines.

HSV is a double-stranded DNA virus, contains the largest genome (153 kb)among DNA viruses that proliferate in nuclei, which encodes 84 kinds ofopen reading frames. The genome consists of the L (long) and S (short)components, each having a unique sequence flanked by inverted repeatsequences on its both sides. The complete nucleotide sequence of theviral genome has been determined and the functions of almost all viralgenes have been elucidated. Martuza et al. developed herpes simplexvirus mutant G207, a multi-gene mutant of herpes simplex virus type 1(HSV-1) with a deletion in the γ34.5 gene and a lacZ gene insertion inthe ICP6 gene (refer to e.g., non-patent literature 5). G207 is superiorto other virus vectors from the therapeutic viewpoint. G207 isreplicated in dividing cells, thereby causing lysis and death ofinfected cells, whereas in non-dividing cells, the virus proliferationis markedly weak. Inoculation with G207 into tumors established inathymia mice suppressed the tumor growth due to tumor-specificreplication and prolonged the survival period of tumor-bearing mice(refer to e.g., non-patent literature 6). Further, in immune responsivemice, intratumoral inoculation with G207 induces tumor-specific immuneresponses, thereby suppressing the growth of tumors that have not beeninoculated with G207 as well (refer to e.g., non-patent literature 7).In this case, G207 is acting as an in situ cancer vaccine (refer toe.g., patent literature 1). To date, gene therapy using herpes simplexvirus mutant G207 has been performed, focusing on brain tumors, andtheir clinical application has also started in the U.S. (refer to e.g.,non-patent literature 8).

The murine colon carcinoma cell line CT26 is poorly immunogenic and doesnot induce tumor-specific CTLs at the detectable level. CT26 is widelyused as a syngeneic tumor model to study immunotherapy (refer to e.g.,non-patent literatures 9 and 10). As a tumor-specific antigen in CT26,the MHC class I-restricted AH1 peptide, derived from an envelop protein(gp70) of an endogenous murine leukemia virus, was identified (refer toe.g., non-patent literature 10). It was confirmed that CT26 tumors whichhave been subcutaneously established can be treated by adoptive immunitycell transfer of peptide-specific CTLs and that there is a correlationbetween induction of tumor-specific CTLs and antitumor effects (refer toe.g., non-patent literature 10). To investigate the efficacy of HSV-1mutant G207 as an in situ cancer vaccine, the inventors have used thepoorly-immunogenic murine colon carcinoma cell line CT26, whichexpresses the tumor antigen identified. Further, the inventors haveevaluated the efficacy of G207 using the syngeneic M3 mouse melanomamodel to clarify that antitumor responses induced by intratumoralinoculation with G207 can be commonly used (refer to e.g., non-patentliteratures 11 and 12).

On the other hand, following methods for inactivating viruses are known:(a) physical inactivation methods such as heat treatment (refer to e.g.,non-patent literature 13), ultraviolet irradiation (refer to e.g.,patent literature 2), γ-irradiation (refer to e.g., patent literature3), electron beam irradiation (refer to e.g., patent literatures 4 and5), pressure treatment (refer to e.g., patent literature 6), andenergizing treatment (refer to e.g., patent literature 7); (b) chemicalinactivation methods such as sterilization using phenol, formalin,alcohol, etc., alkaline treatment (refer to e.g., patent literature 8),contact with singlet oxygen, which consists of normal oxygen moleculesexcited electronically and being at the high state in energy (refer toe.g., patent literature 9), and deoxyribonuclease treatment; and (c)thecombination of these physical inactivation methods and chemicalinactivation methods (refer to e.g., patent literatures 10 and 11).

In addition, the following are described regarding HSVgD as vaccinesagainst herpes viruses: methods of producing recombinant HSVgD (refer toe.g., patent literature 12), recombinant HSVgD vaccine (refer to e.g.,patent literature 13), recombinant DNA encoding HSV-2gD and the protein(refer to e.g., patent literature 14), vaccine formulations consistingof HSVgD and 3-deacylated monophosphoryl lipid A (refer to e.g., patentliterature 15), methods of producing recombinant HSVgD using insectcells (refer to e.g., patent literature 16), the HSVgD moleculeconsisting of 300 amino acid sequences (refer to e.g., patent literature17), a vaccine composition containing HSVgD or an HBV antigen inconjunction with an adjuvant (refer to e.g., patent literature 18), anda fusion protein of HSVgB polypeptide and HSVgD (refer to e.g., patentliterature 19). However, nothing has been known of using HSVgD for acancer vaccine.

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Metastasis of cancer is a pathologic condition that is extremelydifficult to treat; there have been no effective treatment to date.Although it has been reported that some limited number ofchemotherapeutic drugs are efficacious, their side effects are regardedas questionable. In spite of recent drastic advancements in gene therapyusing virus vectors, there are serious problems in terms of safety. Anobject of the present invention is to provide an antitumor agent, atumor immunity inducer, a T cell activator, and a dendritic cellactivator, which are extremely safe and capable of inducing an antitumorimmune reaction enabling immunotherapy for a cancer in such a way thatan antitumor effect on a human distant tumor such as a metastatic tumoris exhibited. Another object of the present invention is to provide,using such an agent, inducer, or activator, enhancement of an antitumoreffect, a method for treating a distant tumor such as a metastatictumor, a method for inducing tumor immunity, a method for identifying atumor antigen, a method for activating T cells, and a method foractivating dendritic cells.

The inventors have enthusiastically studied to solve the above-mentionedproblems. Considering, in putting cancer therapy by virus vectors intopractical use, safety in their use is a prerequisite, they prepared theinactivated HSV that was completely devoid of infectivity and has virusDNA destroyed, by subjecting the wild- type HSV to ultravioletirradiation and heat treatment. They inoculated this inactivated HSVdirectly into malignant tumor tissues derived from the tumor cell lineCT26 and found that a tumor-specific immune response was induced andmalignant tumors have regressed. They also found that the inactivatedHSV was able to similarly suppress the growth of distant malignanttumors that have not been directly inoculated and that the inactivatedHSV activated human dendritic cells. Likewise, they inoculated HSVgDdirectly into malignant tumor tissues derived from the tumor cell lineCT2 and found that a tumor specific immune response was induced andmalignant tumors regressed and that the growth of distant malignanttumors that have not been directly inoculated was suppressed. They alsofound that HSVgD worked to activate T cells as a costimulatory factorfor T cells and activated human dendritic cells. The present inventionhas been accomplished based on these findings.

DISCLOSURE OF INVENTION

Thus, the present invention relates to the following: An antitumor agentcomprising as an effective ingredient an inactivated herpes simplexvirus or a herpes simplex virus glycoprotein (claim 1); the antitumoragent of claim 1, containing as a main ingredient the herpes simplexvirus that has been inactivated by an ultraviolet treatment and a heattreatment (claim 2); the antitumor agent of claim 1, in which theultraviolet treatment is an irradiation at 4 J/m² for 30 min using a 254nm ultraviolet light (claim 3); the antitumor agent of claim 2, in whichthe heat treatment is a heating at 56° C. for 30 min (claim 4); theantitumor agent of claim 1, in which the herpes simplex virusglycoprotein is herpes simplex virus glycoprotein D (claim 5); theantitumor agent of any one of claims 1 to 5, in which the herpes simplexvirus is the KOS strain of herpes simplex virus type 1 or the 169 strainof herpes simplex virus type 2 (claim 6); a method for treating a tumor,containing administering the antitumor agent of any one of claims 1 to 5directly to the tumor tissue (claim 7); a method for treating a distanttumor, such as a metastatic tumor, containing administering theantitumor agent of any one of claims 1 to 5 directly to the tumor tissue(claim 8); a tumor immunity inducer containing as an effectiveingredient an inactivated herpes simplex virus or a herpes simplex virusglycoprotein (claim 9); the tumor immunity inducer of claim 9,containing as a main ingredient the herpes simplex virus inactivated byan ultraviolet treatment and a heat treatment (claim 10); the tumorimmunity inducer of claim 10, in which the ultraviolet treatment is anirradiation at 4 J/m² for 30 min using a 254 nm ultraviolet light (claim11); the tumor immunity inducer of claim 10, in which the heat treatmentis a heating at 56° C. for 30 min (claim 12); the tumor immunity inducerof claim 9, in which the herpes simplex virus glycoprotein is herpessimplex virus glycoprotein D (claim 13); the tumor immunity inducer ofany of one of claims 9 to 13, in which the herpes simplex virus is theKOS strain of herpes simplex virus type 1 or the 169 strain of herpessimplex virus type 2 (claim 14); a method for inducing tumor immunity,containing inducing a cytotoxic T lymphocytes (CTL) and/or an antibodyreaction by administering the tumor immunity inducer of any one ofclaims 9 to 14 directly to the tumor tissue (claim 15); a method foridentifying a tumor antigen, containing inducing a cytotoxic Tlymphocytes (CTL) and/or an antibody reaction by administering the tumorimmunity inducer of any one of claims 9 to 14 directly to the tumortissue and using the induced cytotoxic T lymphocyte (CTLs) and/orantibody (claim 16); a T cell activator containing as effectiveingredient a inactivated herpes simplex virus or a herpes simplex virusglycoprotein (claim 17); the T cell activator of claim 17, containing asa main ingredient the herpes simplex virus inactivated by an ultraviolettreatment and a heat treatment (claim 18); the T cell activator of claim18, in which the ultraviolet treatment is an irradiation at 4 J/m² for30 min using a 254 nm ultraviolet light (claim 19); the T cell activatorof claim 18, in which the heat treatment is a heating at 56° C. for 30min (claim 20); the T cell activator of claim 17, in which the herpessimplex virus glycoprotein is herpes simplex virus glycoprotein D (claim21); the T cell activator of any one of claims 17 to 21, in which theherpes simplex virus is the KOS strain of herpes simplex virus type 1 orthe 169 strain of herpes simplex virus type 2 (claim 22); a method foractivating a T cell, in which the T cell activator of any one of claims17 to 22 is used (claim 23); a dendritic cell activator, containing asan effective ingredient a inactivated herpes simplex virus or a herpessimplex virus glycoprotein (claim 24); the dendritic cell activator ofclaim 24, containing as a main ingredient the herpes simplex virusinactivated by an ultraviolet treatment and a heat treatment (claim 25);the dendritic cell activator of claim 25, in which the ultraviolettreatment is an irradiation at 4 J/m² for 30 min using a 254 nmultraviolet light (claim 26); the dendritic cell activator of claim 25,in which the heat treatment is a heating at 56° C. for 30 min (claim27); the dendritic cell activator of claim 24, in which the herpessimplex virus glycoprotein is herpes simplex virus glycoprotein D (claim28); the dendritic cell activator of any one of claims 24 to 28, inwhich the herpes simplex virus is the KOS strain of herpes simplex virustype 1 or the 169 strain of herpes simplex virus type 2 (claim 29); anda method for activating a dendritic cell containing administrating thetumor cell activator of any one of claims 24 to 29 directly to the tumortissue (claim 30).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that, compared with the mock-inoculated control group, theintratumoral inoculation with inactivated HSV-1 KOS or inactivated HSV-2169 suppress the growth of the inoculated tumor (right tumor: Rt).(Vertical axis: tumor volume, horizontal axis: days after the start ofvaccine treatment)

FIG. 2 shows that, compared with the mock-inoculated control group, theintratumoral inoculation with inactivated HSV-1 KOS or inactivated HSV-2169 suppresses the growth of a distant tumor (right tumor: Rt).(Vertical axis: tumor volume, horizontal axis: days after the start ofvaccine treatment)

FIG. 3 shows cytotoxic T cell activity induced to CT26 cells byintratumoral inoculation of inactivated HSV-1 KOS.

FIG. 4 shows cytotoxic T cell activity induced to Meth A cells byintratumoral inoculation of inactivated HSV-1 KOS.

FIG. 5 shows effect of enhancing the antigen-presenting capability ofhuman dendritic cells by inactivated HSV. In the FIG. 5, HSV0.1, HSV1and HSV1O represent inactivation of HSV at MOI=0.1, 1, and 10,respectively, and DC: Responder represents the ratio of mature dendriticcells to allo-lymphocytes.

FIG. 6 shows that, compared with the saline-inoculated control group,the intratumoral inoculation with inactivated HSV-1gD suppresses thegrowth of the inoculated tumor (right tumor: Rt) as well as a distanttumor (left tumor: Lt).

FIG. 7 shows that, compared with the saline-inoculated control group,the intratumoral inoculation with inactivated HSV-1gD suppresses thegrowth of the inoculated tumors (right tumor: Rt) as well as a distanttumor (left tumor: Lt) like intratumoral inoculation with inactivatedHSV-1 KOS.

FIG. 8 shows T cell activation by costimulation of anti-CD3 antibody andHSV-1gD.

FIG. 9 shows inhibition of HSV-1gD-induced T cell activation action byan anti-HVEM monoclonal antibody.

FIG. 10 shows an effect of enhancing the antigen-presenting capabilityof human dendritic cells by HSV-1gD. In the FIG. 10, DC: Responderrepresents the ratio of mature dendritic cells to allo-lymphocytes.

FIG. 11 shows human lymphocyte activation by HSVgD.

BEST MODE FOR CARRYING OUT THE INVENTION

The antitumor agents, tumor immunity inducers, T cell activators, anddendritic cell activators according to the present invention are notparticularly limited, as long as they contain as an effective ingredientan inactivated HSV, or an HSV glycoprotein such as herpes simplex virusglycoproteins D (HSVgD), B (HSVgB), or C (HSVgC). The above-mentionedantitumor agents and tumor immunity inducers are extremely useful as insitu cancer vaccines. It is extremely preferable that “in situ” hereinmeans direct inoculation into malignant tumor tissue. Theabove-mentioned inactivation methods can be known virus inactivationmethods including physical inactivation methods such as heat treatment,ultraviolet irradiation, gamma irradiation, electron beam irradiation,pressure treatment, energizing treatment; chemical inactivation methodssuch as sterilization using phenol, formalin, and alcohol, alkalinetreatment, and deoxyribonuclease treatment; and methods combining thesephysical inactivation methods with chemical inactivation methods such asheat treatment in the presence of detergent. Mutagenesis by genemanipulation, however, is not included in inactivation methods herein.

Among these virus inactivation methods, the inactivation methodcombining the treatment of destroying virus DNA (e.g., ultraviolettreatment) with protein denaturation treatment (e.g., heat treatment)that eliminates infectivity is particularly preferable in terms ofsafety of cancer vaccines. Preferably as such UV treatment, anirradiation at 1-10 J/m², particularly at 4 J/m² for 5-60 min, moreparticularly for 30 min, using far-ultraviolet light at a wavelength of190-300 nm having the effect of inducing injury of genes in an organism,particularly ultraviolet light of 254 nm, which is absorbed by DNA andRNA bases and stops replication in cell division by dimers, such asthymine-thymine, thymine-cytosine, cytosine-cytosine, and uracil-uracil,formed by the absorbed light quantum energy, is illustrated. Preferablyas heat treatment, heating at 45-80° C. for 5-10 hours, preferably at56° C. for 30 minutes is illustrated.

Herpes simplex viruses to be inactivated in the production of theantitumor agents, tumor immunity inducers, T cell activators, ordendritic cell activators according to the present invention includeHSV-1 and herpes simplex virus type 2 (HSV-2) wild strains as well asmutant strains mutagenized by gene manipulation. They are preferablycompletely free of anything harmful after inactivation treatment. Onespecific preferable example is the wild-type HSV, such as, for example,the KOS strain of HSV-1 or the 169 strain of HSV-2. Further,aforementioned HSV glycoproteins such as HSVgD, HSVgB, and HSVgC includeHSV glycoproteins such as gD (glycoprotein D), gB (glycoprotein B), gC(glycoprotein C), etc. derived from mutant strains mutagenized by genemanipulation and inactivated strains, in addition to HSV-1 and HSV-2wild-type strains. These HSV glycoproteins can preferably be produced asrecombinant proteins, using bacteria (e.g., E. coli), yeasts, insectcells, mammalian cells, etc. as host cells.

The antitumor agents and tumor immunity inducers according to thepresent invention are useful as a preventive drug or a therapeutic agentfor recurrence and metastasis of cancers such as malignant brain tumor,serving as an in situ cancer vaccine. T cell activators and dendriticcell activators according to the present invention are useful as apreventive drug and a therapeutic agent for recurrence and metastasis ofcancers such as malignant brain tumors as well as for various otherdiseases that require activation of T cells and that of dendritic cells(enhancement of antigen-presenting capability etc.). When such agents,inducers, or activators are used as pharmaceuticals, various mixingingredients for preparation, such as a pharmaceutically acceptablecommon carrier, a binder, a stabilizer, an excipient, a diluent, abuffer, a disintegrator, a solubilizer, a solubilizing agent, and atension agent can be added. Such preventative or therapeutic agents canbe administered orally or parenterally. That is, they may beadministered orally in commonly used administration forms such as, forexample, powders, granules, capsules, syrups, and suspensions, orparenterally in the form of an injection in dose forms such as asolution, an emulsion, and a suspension. The agents may also beadministered in a spray form into the nostrils. However, it ispreferable to inoculate them directly to malignant tumor tissue in thatimmediate tumor specific immune responses can be induced.

The methods for treating tumors according to the present invention,preferably the ones for treating distant tumors such as metastatictumors are not particularly limited, as long as they are the treatmentmethod in which an antitumor agent according to the present invention isadministered by direct inoculation to a tumor tissue. Such therapeuticmethods enable treatment and prevention of recurrence and metastatic ofmalignant tumors. The methods for inducing immune responses according tothe present invention are not particularly limited as long as they arethe method for inducing CTLs and/or an antibody reaction byadministering the tumor immunity inducer according to the presentinvention directly to a tumor tissue. The use of the methods forinducing tumor immunity according to the present invention make itpossible to investigate the mechanism of action of immune responseinduction, especially to a distant tumor by direct inoculation to amouse and other tumor model. In addition, induction of CTLs and anantibody reactions to the living body is also possible. The methods foridentifying tumor antigens according to the present invention are notparticularly limited, as long as they are the method for inducing CTLsand/or an antibody reaction inside the living body byadministering/inoculating a tumor immunity inducer according to thepresent invention directly to/into a mouse or other animal tumor model,and using the induced CTLs and/or the induced antibody. For example,identification of a new tumor antigen is enabled by performing genetransfer of a cDNA library into cells, co-culturing of mammalian cellsthat have expressed proteins encoded by the cDNAs together with CTLsinduced, and analyzing the cDNA encoding the protein that the peptiderecognized by the CTLs is originated.

The methods for activating T cells according to the present inventionare not particularly limited, as long as they are the method foractivating human or other animal T cells in vivo, in vitro, or ex vivo,using a T cell activator according to the present invention. However, inactivating T cells in vivo, it is preferable to administer the T cellactivator of the present invention directly to a tumor tissue. Themethods for activating dendritic cells according to the presentinvention are not particularly limited, as long as they are the methodfor activating human or other mammalian dendritic cells in vivo, invitro, or ex vivo, using a dendritic cell activator according to thepresent invention. However, in activating dendritic cells in vivo, it ispreferable to administer the dendritic cell activator of the presentinvention directly to a tumor tissue. The use of the method foractivating T cells and the method for activating dendritic cellsaccording to the present invention enables treatment and research ofdiseases requiring activation of T cells and/or dendritic cells.

The present invention is explained with the following examples in moredetail, but the technical scope of the present invention is not limitedby these examples.

EXAMPLE 1 Viruses, Cell Lines, and Proliferation

As viruses, the KOS strain of HSV-1 and the 169 strain of HSV-2(provided by Dr. Yoshiko Seto) were used. African green monkey kidneyVero cells (purchased from ATCC) were used for proliferation of HSVs.Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% heat-inactivated fetal calf serum (IFCS), and inDMEM supplemented with 1% IFCS when viruses were proliferating. Afterrepeating freeze-thaw of the cells in the virus buffer (150 mM NaCl-20mM Tris pH7.5) and ultrasonicating the extract, viruses were recoveredfrom the supernatant.

EXAMPLE 2 Inactivation of HSV

Test HSVs were subjected to inactivation treatment by ultraviolettreatment and heat treatment. The infection efficiency of the HSVs usedbefore inactivation was 2×10⁸ plaque-formation units/ml. As inactivationby ultraviolet rays, the viruses were irradiated at 4 J/m² for 30 minusing 254 nm ultraviolet light on ice, according to the partly modifiedmethod of David C. J. et al. (J. virology, 62, 4605-4612, 1988).Subsequently, the UV-treated HSVs were subjected to heat treatment. Asinactivation by heat treatment, thermal denaturation treatment of theHSVs were performed at 56° C. for 30 min according to the partlymodified method of Hitsumoto Y et al. (Microbiol. Immunol., 27, 757-765,1983).

EXAMPLE 3 In Situ Cancer Vaccine in a Mouse Bilateral Subcutaneous TumorModel Using Inactivated HSV

Six to eight-week old female BALB/c mice were anesthetized (with 0.25 mlof anesthetic liquid: 84% saline, 10% pentobarbital [50 mg/ml], 6%ethanol), and then 5×10⁵ CT26 cells /100 μl was subcutaneously implantedbilaterally into mice. Treatment started when the diameter of the tumorreached about 5 mm. The extract of Vero cells used for virus preparationwas UV-irradiated and heat-treated, and used as a control (hereinaftercalled “mock”). Either inactivated HSV or mock was administered only tothe right tumor on days 0 and 3 after the start of treatment.Thereafter, the volumes of both of the tumors were periodicallymeasured. Volume was calculated as major axis×(minor axis)²÷2. FIG. 1shows the result of the periodical changes in tumor volume of the righttumor (Rt), into which CT 26 was directly administered (inoculated).FIG. 2 shows the result of the periodical changes in tumor volume of theleft tumor (Lt), which was a distant tumor. As observed in FIG. 1, inthe inactivated HSV-1 KOS group (n=7) or the inactivated HSV-2 169 group(n=7), the growth of CT26 tumor was statistically significantlysuppressed 15 days after the start of administration as compared withthe mock group (n=6). FIG. 2 shows a similar result on the left distanttumor, to which inactivated HSV had not directly been administered. Inthe inactivated HSV-1 KOS group or the inactivated HSV-2 169 group, thegrowth of CT26 tumor was statistically significantly suppressed 15 daysafter the start of administration as compared with the mock group. Theseresults indicated that the in situ cancer vaccine according to thepresent invention exhibits the antitumor effects on distant tumors aswell.

EXAMPLE 4 Cytotoxic T Cell Activity Test with Inactivated HSV

Spleen cells were isolated from the mice that had received an in situcancer vaccine (inactivated HSV-1 KOS strain) on day 10 after the startof treatment and were co-cultured with CT26 cells for seven days, andthen ⁵¹Cr release test was performed. Either CT26 cells or Meth A cellsinto which 50 μCi of ⁵¹Cr had been incorporated were used as targetcells. A total of 2.5×10³ target cells, together with 4.0×10⁴, 1.3×10⁴,or 4.3×10³ spleen cells as effector cells, were incubated at 37° C. for4 hours in the presence of 5% carbon dioxide in a well of 96-wellplates. ⁵¹Cr emitted into the culture supernatant was measured with agamma counter. FIG. 3 and FIG. 4 show the result of the lysis rate ofCT26 cells (n=3) Meth A cells (n=3), respectively, in various effectorcell/target cell ratios (E/T ratios). As observed from FIG. 3, in the insitu cancer vaccine group, antitumor CTL activity was large comparedwith the mock group, and the larger the E/T ratio, the larger thedifference of the antitumor CTL activity was. On the other hand, no CTLactivity was induced in Meth A cells, either in the in situ cancervaccine group or in the mock group. These results support the fact thatthe in situ cancer vaccine according to the present invention induces aspecific tumor CTL activity.

EXAMPLE 5 Enhancement Effect of Antigen-Presenting Ability of HumanDendritic Cells by Inactivated HSV

Immature dendritic cells were obtained from human peripheral blood byseparating and culturing a CD14-positive subset by the immunomagneticbead method. Specifically, a magnetic bead-coupled monoclonal antibody(20 μl/10⁷ cells; manufactured by Miltenyi Biotec) against CD14 antigenwas incubated with the cells at 4° C. for 15 min. The CD14-positivefraction was isolated by magnetically separating bead-bound cells usinga magnetic cell separation system (MACS). The fraction was plated at aconcentration of 1×10⁶ cells/well and cultured for 6-7 days in RPMImedium containing 10% FBS (fetal bovine serum; Gb4) prepared to have 100ng/ml of GM-CSF (manufactured by IBL Co., Ltd. ) and IL-4 (IBL) each.Immature dendritic cells were thus obtained. After confirming theexpression of surface antigens (CD1a+, CD14−, CD80+, and CD86+) asimmature dendritic cells with a flow cytometer, the inactivated HSV-1KOS strain prepared by the method described in Example 2 was added tothe medium at concentrations of multiplicity of infection (MOI)=0.1, 1,and 10 plaque-forming units (PUFs) /cells, which had been measuredbefore inactivation, or alternatively mock was added instead of thevirus, maturation factors TNF-α (manufactured by IBL Co., Ltd.) andIFN-γ (manufactured by IBL Co., Ltd.) were added 1 hour later at thefinal concentration of 10 ng/ml and 100 ng/ml, respectively, and thecells were incubated overnight. On the next day, the expression of thesurface antigen CD83 (which was negative in immature dendritic cells) asmature dendritic cells was confirmed with a flow cytometer.

Subsequently, the mature dendritic cells induced with the addition ofthe inactivated HSV-1 KOS strain were examined for their ability toactivate allo-lymphocytes. Mature dendritic cells induced by theaddition of TNF-α (10 ng/ml) and IFN-γ (100 ng/ml) alone, without mockor inactivated HSV, were used as a control. The CD14-negative fractionof peripheral blood at a concentration of 6×10⁴ cells asallo-lymphocytes (responders) and mature dendritic cells (DCs) asstimulators whose proliferation had been stopped by irradiation (15Gy)were co-cultured (the ratio of DCs to responders was 1:20 or 1:40) for 3days. The cultured lymphocytes were labeled with ³H thymidine at 0.027Mbq/well for about 12 hours and the amount (CPM: counter per minute) of³H-thymidine incorporated was measured using the top counter. FIG. 5shows the relative amount of ³H-thymidine incorporated by lymphocytesafter being co-cultured with mature dendritic cells to that of thecontrol. As a result, it was indicated that mature dendritic cells towhich the inactivated HSV-1 KOS strain was added have a higher abilityto activate lymphocytes as compared with the control or inactivatedmock. It was clarified by these results that the inactivated HSV has aneffect of enhancing the antigen-presenting ability of human dendriticcells.

EXAMPLE 6 In Situ Cancer Vaccine in a Mouse Bilateral Subcutaneous TumorModel Using HSVgD

Six to eight-week old female BALB/c mice were anesthetized (with 0.25 mlof anesthetic liquid: 84% saline, 10% pentobarbital [50 mg/ml], 6%ethanol), and then 5×10⁵ cells/100 μl of CT26 was subcutaneouslyimplanted bilaterally into mice. Treatment started when the diameter ofthe tumor reached about 5 mm. Saline was used as a control. Eithersaline solution of HSV type 1 (HSV-1) gD protein (manufactured byViroStat Inc.) or saline was administered (30 μl each) only to the righttumor on days 0 , 3, 6, and 9 after the start of treatment. Thereafter,the volumes of both of the tumors were periodically measured. Volume wascalculated as major axis×(minor axis)²÷2. FIG. 6 shows the result of theperiodical changes in tumor volume of the right tumor (R) into whichHSVgD was directly administered (inoculated), together with the resultof the periodical changes in tumor volume of the left tumor (L) whichwas a distant tumor. As observed from FIG. 6 (right), in the HSVgD totaldose (30 ng) group (n=7), the growth of CT26 tumors on the administeredside was statistically significantly suppressed 24 days after the startof administration as compared with the saline solution group (n=3). FIG.6 (left) indicated a similar result on the left distant tumor: In theHSVgD-1 3 ng group and the 30 ng group, the growth of CT26 tumors wasstatistically significantly suppressed 24 days after the start ofadministration as compared with the mock group. These results revealedthat the in situ cancer vaccine according to the present inventionexhibits an antitumor effect on distant tumors as well. Further, theHSVg antitumor effect was observed to depend on its dosage.

To reconfirm the antitumor effect of the in situ cancer vaccine usingaforementioned HSVgD, BSA (manufactured by Sigma Chemical Corporation)was used as a negative control and inactivated HSV-1 KOS strain preparedin a similar manner to example 2 as a positive control. HSVgD or BSAdissolved in saline was administered (30 μl each) only to the righttumor on days 0, 3, and 6 after the start of the treatment. Thereafter,the volumes of both of the tumors were measured periodically.Inactivated HSV-1 KOS strain was administered (50 μl) only to the righttumor on day 0 and 3 after the start of the treatment. FIG. 7 shows aresult of the periodical changes of the tumor volume of the right tumor(R), into which <HSVgD> was directly administered (inoculated), togetherwith a result of the periodical changes of the tumor volume of the lefttumor (L), which was a distant tumor. As observed from FIG. 7 (right),the HSVgD total dose (30 ng) (n=7) exhibited a behavior similar to thatexhibited by the KOS group (the infection efficiency beforeinactivation: 2×107 PFU) (n=7). Eighteen days after the start of theadministration, the growth of CT26 tumors on the administration side wassuppressed as compared with the BSA total dose (300 ng) group (n=6). Asobserved from FIG. 7 (left), as for the left distant tumor to whichHSVgD had not directly been administered, the HSVgD 30 ng group (n=8)exhibited a behavior similar to that exhibited by the KOS group (n=7).Eighteen days after the start of administration, the growth of CT26tumors was statistically significantly suppressed as compared with theBSA total dose (300 ng) group (n=6). It was reconfirmed that the in situcancer vaccine according to the present invention exhibits antitumoreffects on distant tumors as well.

EXAMPLE 7 Effect of HSVgD on T Cells

T cells were obtained from the lymph node of 6-week-old female C57BL/6mice by the immunomagnetic bead method. Specifically, magneticbead-coupled monoclonal antibodies (10 μl/10⁷ cells each; manufacturedby Miltenyi Biotec) against CD90, CD45R, MHC ClassII, and CD11c wereincubated with the cells at 4° C. for 15 min. The T cell fraction wasisolated by magnetically separating bead-bound cells using a magneticcell separation system (MACS; manufactured by Miltenyi Biotec). Thefraction (CD90+, CD45R−, MHC ClassII−, and CD11c−) was finally isolatedas T cells. T cells (6×10⁴ cells/well) were cultured for 5 days in thepresence or absence of anti-CD3 antibody (0.01 ng/ml) and/or HSV-1 gDprotein (0.05 ng/ml; manufactured by ViroStat Inc.). After labeling thecultured lymphocytes with ³H-thymidine for about 12 hours, the amount(CPM) of ³H-thymidine incorporated was measured by the top counter. Theresult is shown in FIG. 8. As observed from FIG. 8, it was revealed thatT cells are not activated by either of the anti-CD3 antibody thatstimulates the T cell receptor or HSV-1 gD that is a ligand forherpes-virus entry mediator (HVEM), but can be activated only when theyare stimulated by both HSVgD and the anti-CD3 antibody. It has beenfound that LIGHT, a member of TNF cytokine family, activates T cells viaHVEM as a costimulatory factor (Nat Med 6, 283-289, 2000). Being aligand for HVEM, HSVgD has been revealed to work for activation of Tcells by acting as a costimulatory factor to T cells.

EXAMPLE 8 Activation of T Cells by HSVgD Via HVEM

Mouse T cells were separated using the same method as that used inExample 7. T cells were plated at 6×10⁴ cells/well and cultured for 5days in the presence or absence (control) of HSV-1 gD (0.2 or 0.4 ng)and/or anti-HVEM monoclonal antibody (0.01 ng/ml; provided by DrPatricia G. Spear, Northwestern University, USA). The culturedlymphocytes were labeled with ³H-thymidine for about 24 hours and theamount (CPM) of ³H-thymidine incorporated was measured using the topcounter. The result is shown in FIG. 9. As observed from FIG. 9,although T cells to which 0.4 ng of HSVgD had been added were activated,the activation of T cells was inhibited by the addition of anti-HVEMmonoclonal antibody, which blocks HVEM regarded as the receptor forHSVgD, to 0.4 ng of HSVgD. This experiment demonstrated that activationof T cells by HSVgD results from the signaling via the HVEM receptor.

EXAMPLE 9 Enhancement Effect of Antigen-Presenting Ability of HumanDendritic Cells by HSVgD

Immature dendritic cells were obtained from human peripheral blood onday 7 of culture by a method basically similar to that used in Example 5and supplemented with a medium (control), HSVgD (0.2 ng/ml or 0.4ng/ml), or mock. This experiment examined the ability of immaturedendritic cells with HSVgD (0.2 ng or 0.4 ng) added to them to activateallo-lymphocytes. The CD14-negative fraction of peripheral blood at aconcentration of 6×10⁴ cells as allo-lymphocytes (responders) and maturedendritic cells (DCs) as stimulators whose proliferation had beenstopped by irradiation (15Gy) were co-cultured (the ratio of DCs toresponders is 1:20 or 1:40) for 5 days. The cultured lymphocytes werelabeled with ³H-thymidine at 0.027 Mbq/well for about 12 hours and theamount (CPM: counter per minute) of ³H-thymidine incorporated wasmeasured using the top counter. FIG. 10 shows the relative amount of³H-thymidine incorporated by lymphocytes to that of the control afterbeing co-cultured with dendritic cells. As a result, dendritic cells towhich HSVgD was added have a higher ability to activate lymphocytes ascompared with the control or mock. These results clarified that HSVgDhas an effect of enhancing the antigen-presenting ability of humandendritic cells.

EXAMPLE 10 Activation of Human Lymphocytes by HSVgD

The CD14-negative fraction of peripheral blood was obtained byseparation and removal of the CD14-positive subset from human peripheralblood by the immunomagnetic bead method. Specifically, a magneticbead-coupled monoclonal antibody (20 μl/10⁷ cells) against CD14 antigenwas incubated with the cells at 4° C. for 15 min. The CD14-negativefraction of peripheral blood was obtained by magnetic separation andremoval of bead-bound cells using a magnetic cell separation system(MACS). Lymphocytes in this peripheral blood were plated at 6×10⁴cells/well as allo-lymphocytes (responder), supplemented with OKT3 (0 or10 ng/ml) as a stimulatory molecule of T cell receptors, and further,with either the inactivated HSV-1 KOS strain (infection efficiencybefore inactivation: 2×10⁶PFU), gD (0.2 ng/ml), or mock (10 μl) as acostimulatory molecule, and cultured. On day 6 of culture, the culturedlymphocytes were labeled with ³H-thymidine (0.027 Mbq/well) for about 12hours, and then the amount (CPM) of ³H-thymidine incorporated wasmeasured by the top counter. The result is shown in FIG. 11. Theseresults indicated that T cells are not activated in the absence of ananti-CD3 monoclonal antibody OKT3 and that human lymphocytes can beactivated in the co-presence of gD and OKT3 (10 ng/ml). It can be thusconsidered that, like the experiment with mice (Example 7), HSVgD canactivate T cells as a costimulatory factor in humans as well.

INDUSTRIAL APPLICABILITY

According to the present invention, antitumor agents, tumor immunityinducers, T cell activators, and dendritic cell activators, extremelysafe and capable of inducing an antitumor immune reaction enablingimmunotherapy for cancer in such a way that the antitumor effect on ahuman distant tumor such as metastatic tumors can be exerted. Theseantitumor agents, tumor immunity inducers, etc. are expected to havebroad applications to cancer therapy.

1. An antitumor agent comprising a herpes simplex virus glycoprotein asan effective ingredient. 2-4. (canceled)
 5. The antitumor agent of claim1, wherein the herpes simplex virus glycoprotein is herpes simplex virusglycoprotein D.
 6. (canceled)
 7. A method for treating a tumor,comprising administering the antitumor agent of claim 1 directly to atumor tissue.
 8. A method for treating a distant tumor, such as ametastatic tumor, comprising administering the antitumor agent of claim1 directly to a tumor tissue.
 9. A tumor immunity inducer comprising aherpes simplex virus glycoprotein as an effective ingredient. 10-12.(canceled)
 13. The tumor immunity inducer of claim 9, wherein the herpessimplex virus glycoprotein is herpes simplex virus glycoprotein D. 14.(canceled)
 15. A method for inducing tumor immunity, comprising inducingcytotoxic T lymphocytes (CTL) and/or an antibody reaction by directlyadministering the tumor immunity inducer of claim 9 to a tumor tissue.16. A method for identifying a tumor antigen, comprising inducing acytotoxic T lymphocytes (CTL) and/or an antibody reaction byadministering the tumor immunity inducer of claim 9 directly to a tumortissue and using the induced cytotoxic T lymphocyte (CTLs) and/orantibody reaction.
 17. A T cell activator comprising a herpes simplexvirus glycoprotein as effective ingredient. 18-20. (canceled)
 21. The Tcell activator of claim 17, wherein the herpes simplex virusglycoprotein is herpes simplex virus glycoprotein D.
 22. (canceled) 23.A method for activating a T cell, wherein the T cell activator of claim17 is used.
 24. A dendritic cell activator, comprising a herpes simplexvirus glycoprotein as an effective ingredient. 25-27. (canceled)
 28. Thedendritic cell activator of claim 24, wherein the herpes simplex virusglycoprotein is herpes simplex virus glycoprotein D.
 29. (canceled) 30.A method for activating a dendritic cell comprising administrating thedendritic cell activator of claim 24 directly to a tumor tissue.